Simultaneous identification of 78 components in Aloe barbadensis Mill.

Abstract

The present application relates to a kind of method for simultaneously identifying 78 components in traditional Chinese medicine aloe barbadensis miller, using HPLC-DAD-ESI-IT-TOF-MS n The present application detects traditional Chinese medicine aloe barbadensis miller through specific mass spectrometry conditions and specific gradient elution program, so that the present application can simultaneously identify 78 components. In addition, in addition to the index components such as aloe-emodin, aloin D and aloin A, the present application can also simultaneously identify 14 compounds with pharmacological activity, which can more comprehensively and completely reflect the quality of traditional Chinese medicine aloe barbadensis miller, and is of great significance for the quality control of traditional Chinese medicine aloe barbadensis miller. Especially nowadays, the quality of traditional Chinese medicine aloe barbadensis miller on the market is uneven, and simultaneously identifying multiple index components with pharmacological activity is more important for quality evaluation. At the same time, the method of the present application has good repeatability and stability.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine quality control, and relates to a method for simultaneously identifying 78 components in traditional Chinese medicine Aloe vera. BACKGROUND

[0002] Traditional Chinese medicine aloe is the juice concentrate dry matter of leaves of Aloe vera (L.) Burm., A. ferox Miller or other related plants in the same genus, wherein Aloe vera is most widely used. Aloe vera has the effects of purgation, clearing liver and purging fire, and killing insects and treating malnutrition, and can be used for treating constipation, convulsions and the like. Aloe and its main component aloin have pharmacological activities such as bacteriostasis, anti-tumor, anti-inflammatory and purgation, so that aloin is currently used as an index component for quality evaluation. The 2020 edition of Chinese Pharmacopoeia also only establishes the quality control index of aloin. However, the types of chemical components in aloe are various, and only using a single component as an index component of traditional Chinese medicine aloe is not enough to comprehensively and completely reflect the quality of traditional Chinese medicine aloe. Especially, the quality of traditional Chinese medicine aloe on the market is uneven, and different origins, producing areas and processing technologies can all affect the quality of traditional Chinese medicine aloe.

[0003] Therefore, developing a method capable of simultaneously identifying more active index substances in traditional Chinese medicine Aloe vera (juice concentrate dry matter of leaves of Aloe vera plant) to comprehensively and completely reflect the quality of traditional Chinese medicine Aloe vera has become a problem to be solved by those skilled in the art. SUMMARY

[0004] To solve the problems in the prior art, the application provides a method for simultaneously identifying 78 components in traditional Chinese medicine Aloe vera, and the technical scheme adopted by the application is as follows:

[0005] The method comprises the following steps

[0006] (1) Preparation of a test sample solution: a traditional Chinese medicine Aloe vera powder sample is taken, methanol is added, and ultrasonic extraction is performed; the supernatant is taken after centrifugation, filtration is performed, the filtrate is subjected to nitrogen blowing, and methanol is used for dissolution to obtain a test sample solution;

[0007] (2) Preparation of a control sample solution: a control sample is dissolved in methanol to obtain a control sample solution;

[0008] (3) Detection: the test sample solution of step (1) and the control sample solution of step (2) are injected into an HPLC-DAD-ESI-IT-TOF-MS n for detection;

[0009] The elution mode of the HPLC-DAD is gradient elution, the mobile phase of the gradient elution comprises mobile phase A and mobile phase B; the mobile phase A is pure water, and the mobile phase B is acetonitrile; the sum of the volume fractions of the mobile phase A and the mobile phase B is 100%; the elution procedure of the gradient elution is as follows:

[0010] Time Mobile phase A / vol% Mobile phase B / vol% 0 - 45 min 80％～90％ 10％～20％ 45 - 80 min 75％～80％ 20％～25％ 80 - 120 min 70％～75％ 25％～30％ 120 - 140 min 65％～70％ 30％～35％ 140 - 150 min 50％～65％ 35％～50％ 150 - 160 min 30％～50％ 50％～70％ 160 - 170 min 0％～30％ 70％～100％ 170 - 175 min 0％ 100％

[0011] Further, in step (3),

[0012] The ion source is an electrospray ion source (ESI); and / or

[0013] The detection mode is a negative ion mode; and / or

[0014] The mass scan range is m / z 50-1000 (MS), m / z 50-1000 (MS 2 and MS 3 ); and / or

[0015] The relative collision-induced dissociation energy is 50%; and / or

[0016] The heating block temperature is 200°C; and / or

[0017] The curved desolvation tube temperature is 200°C; and / or

[0018] The detection voltage is 1.70 kV; and / or

[0019] The interface voltage is (+) 4.5 kV, (-) -3.5 kV; and / or

[0020] The nebulization gas flow rate is 1.5 L / min.

[0021] Further, in step (1),

[0022] The ultrasonic temperature is 20-50°C, and the ultrasonic time is 20-60 min; and / or

[0023] The filtration is microfiltration membrane filtration, and the pore size of the microfiltration membrane is 0.20-0.22 μm; and / or

[0024] The concentration of the test sample solution is 5-20 mg / mL.

[0025] Further, in step (2),

[0026] The control is aloeemodin, aloin A, aloeemodin, 7-O-methyl aloeemodin A, aloin, emodin methyl ether, altechromone A, emodin-8-O-β-D-glucopyranoside and aloeemodin A; and / or

[0027] The concentration of the control solution is 0.2-2 mg / mL.

[0028] Further, in step (3),

[0029] The chromatographic column is Phenomenex Gemini C 18 The chromatographic column has a size of 250 mm*4.6 mm, and the chromatographic column filler has a particle size of 5 μm; and / or

[0030] The guard column is Phenomenex Security Guard, and has a size of 4 mm*3.0 mm, and the guard column filler has a particle size of 5.0 μm; and / or

[0031] The column temperature is 35 °C; and / or

[0032] The injection volume is 10 μL; and / or

[0033] The wavelength is 190-400 nm; and / or

[0034] The flow rate is 0.7-1.3 mL / min.

[0035] Compared with the prior art, the present application has the following beneficial effects:

[0036] The present application adopts the liquid chromatography-diode array detector-electrospray ionization-ion trap-time-of-flight-mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS n ) technology to detect the Chinese medicine Aloe barbadensis Miller, through specific mass spectrometry conditions and specific gradient elution procedures, so that the present application can simultaneously identify 78 components. In addition, in addition to the index components such as aloe-emodin, aloin D, and aloin A, the present application can also simultaneously identify 14 compounds with pharmacological activities, which can more comprehensively and completely reflect the quality of the Chinese medicine Aloe barbadensis Miller, and has important significance for the quality control of the Chinese medicine Aloe barbadensis Miller. Especially, the quality of the Chinese medicine aloe on the market is uneven, and the simultaneous identification of a plurality of index components with pharmacological activities is more important for quality evaluation. At the same time, the method of the present application has good repeatability and stability. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Figure 1 is a base peak chromatogram of the Chinese medicine Aloe barbadensis Miller in the negative ion detection mode at 0-40 min according to the present application;

[0038] Figure 2 Figure 2 is a base peak chromatogram of the Chinese medicine Aloe barbadensis Miller in the negative ion detection mode at 40-80 min according to the present application;

[0039] Figure 3The base peak chromatogram of Aloe barbadensis Miller in the negative ion detection mode at 80-120 min in Example 1 of the present application;

[0040] Figure 4 The base peak chromatogram of Aloe barbadensis Miller in the negative ion detection mode at 120-170 min in Example 1 of the present application. DETAILED DESCRIPTION

[0041] It is necessary to point out that the following examples are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Some non-essential improvements and adjustments to the present application made by those skilled in the art based on the content of the present application still fall within the scope of protection of the present application.

[0042] The raw materials used in the examples are all conventional commercially available raw materials. The instruments used in the examples are all conventional commercially available instruments. Among them, the sample for preparing the test solution is aloe powder, which is purchased from Shaanxi Jiahe Pharmaceutical Co., Ltd. The aloe powder is a concentrated and dried juice of Aloe barbadensis Miller leaves, and the aloe glycoside content is ≥18%, and the ash content is less than 2%. Aloe new glycoside D (PS0801-0025), aloe substance A (PS0136-0025), aloe barbadensis (PS1255-0025), 7-O-methyl aloe new glycoside A (PS0799-0010), emodin methyl ether (PS010626), and emodin-8-O-β-D-glucopyranoside (PS011865) are all purchased from Chengdu Pusai Biological Technology Co., Ltd. Aloe bitter (B23136-20) is purchased from Shanghai Yuanye Biological Technology Co., Ltd. Aloe chromone A (M095312-5mg) and aloe ning A (M044622-5mg) are both purchased from Beijing Mayride Technology Co., Ltd. The purity of all the control samples is greater than 98.0% (HPLC, 254 nm).

[0043] It should be noted that the volume fraction range in the gradient elution program, such as the volume fraction of mobile phase A being 80%-90%, represents that the volume fraction of mobile phase A is uniformly increased from 80% to 90% in this time period. The atomizing gas in the mass spectrometry conditions is nitrogen.

[0044] Example 1 The present example relates to the detection method of the present application

[0045] (1) Preparation of test solution

[0046] Take aloe powder sample, accurately weigh 0.2 g, add 20 mL of methanol, weigh, 20 ℃ ultrasonic extraction for 30 min, cool to room temperature, weigh again, make up the weight loss with methanol, shake well, centrifuge at 8000 rpm for 15 min at 4 ℃, take the supernatant. Take 2 mL of supernatant, filter through a 0.22 μm microporous filter membrane, nitrogen blow to dryness, add 100 μL of methanol to dissolve, and obtain the test solution, store in a -20 ℃ refrigerator for standby.

[0047] (2) Preparation of the control solution

[0048] The control is aloe new glycoside D, aloe glycoside A, aloe rheum, 7-O-methyl aloe new glycoside A, aloe bitter glycoside, emodin methyl ether, altechromone A, emodin-8-O-β-D-glucopyranoside and aloe ning A. Accurately weigh 10.00 mg of the control, dissolve in 10 mL of methanol to prepare a 1 mg / mL solution, accurately pipette 100 μL of each control solution, mix and prepare a mixed control solution containing 9 controls.

[0049] (3) HPLC-DAD-ESI-IT-TOF-MS n Detection

[0050] The test solution of step (1) and the control solution of step (2) are injected into HPLC-DAD-ESI-IT-TOF-MS n for detection, and the chromatographic conditions are as follows:

[0051] Chromatographic column: Phenomenex Gemini C 18 Chromatographic column (250 mm x 4.6 mm, 5 μm);

[0052] Guard column: Phenomenex Security Guard (4 mm x 3.0 mm, 5.0 μm) (Phenomenex, Torrance, CA, USA);

[0053] Column temperature: 35 ℃;

[0054] Injection volume: 10 μL;

[0055] Mobile phase: mobile phase A is pure water, and mobile phase B is acetonitrile;

[0056] Flow rate: 1.0000 mL / min;

[0057] DAD recording wavelength: 190-400 nm.

[0058] The mobile phase gradient elution program is as shown in Table 1:

[0059] Table 1 Gradient elution procedure for Example 1

[0060] Time Mobile phase A / vol% Mobile phase B / vol% 0 - 45 min 80％～90％ 10％～20％ 45 - 80 min 75％～80％ 20％～25％ 80 - 120 min 70％～75％ 25％～30％ 120 - 140 min 65％～70％ 30％～35％ 140 - 150 min 50％～65％ 35％～50％ 150 - 160 min 30％～50％ 50％～70％ 160 - 170 min 0％～30％ 70％～100％ 170 - 175 min 0％ 100％

[0061] Mass spectrometry conditions were as follows:

[0062] Ion source: electrospray ion source (ESI);

[0063] Detection mode: negative ion detection mode;

[0064] Mass scan range: m / z 50-1000 (MS), m / z 50-1000 (MS 2 and MS 3 );

[0065] Relative collision-induced dissociation energy: 50%;

[0066] Heating block temperature: 200°C;

[0067] Curved desolvation tube temperature: 200°C;

[0068] Detection voltage 1.70 kV;

[0069] Interface voltage: (+) 4.5 kV, (-) -3.5 kV;

[0070] Atomization gas (nitrogen) flow rate, 1.5 L / min.

[0071] After detection, the results are shown in Figures 1 to 4 . It should be noted that since the detection time of the present application is 170 min, and 168 chemical components are detected and 78 chemical components are identified, in order to better illustrate the identified components, the detected spectrum is divided into Figures 1 to 4 . Among them, Figure 1 is the base peak chromatogram measured at 0-40 min, Figure 2 is the base peak chromatogram measured at 40-80 min, Figure 3 is the base peak chromatogram measured at 80-120 min, Figure 4 is the base peak chromatogram measured at 120-170 min.

[0072] (4) Data processing and result analysis

[0073] The obtained raw mass spectrum data was analyzed by LCMS Solution v.3.60 software, and the compounds were compared with the reference substances. The retention time, quasi-molecular ion, mass spectrum fragment information and the like of each component in the test sample solution were compared and confirmed with the reference substances. For the compounds without reference substance comparison, the chemical structures were identified according to the retention time, molecular weight, molecular formula, quasi-molecular ion, and reference mass spectrum fragment information reported in the literature, and the SciFinder database was searched. The specific information is shown in Table 2. Specifically, 168 chemical components were simultaneously detected, and 78 chemical components were identified. The 78 identified chemical components are mainly classified into five categories, namely anthraquinones, anthrones, chromones, α-pyrone and others, wherein 3 anthraquinones, 29 anthrones, 35 chromones, 7 α-pyrone and 4 others are identified. Four of the 78 chemical components were identified by reference substance comparison, and the other 74 chemical components were identified by combining mass spectrum data, literature reports and SciFinder database search.

[0074] Table 2 HPLC-DAD-IT-TOF-MS of 78 chemical components in A. barbadense Mill. from the Chinese medicine library n Identification results

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] In addition, according to the literature reports, the 14 compounds C17-C19, C24-C30, C67, C76-C78 all have pharmacological activities. Specifically, the three compounds C76-C78 have potential free radical scavenging activity. C67 has anti-arthritic activity and can be used as an anti-inflammatory agent. C17-C19 has antibacterial activity. C24 can inhibit tyrosinase and can also be used as an active ingredient of aloe medicinal material in the Hundred Scales Shatari Tablet to participate in the multi-component regulation of multiple targets to synergistically inhibit psoriasis inflammation. C25-C27 has anti-inflammatory activity. C28-C30 has the effect of preventing insects. On the basis of being able to identify the index components such as aloe-emodin, aloe new glycoside D and aloe new glycoside A, the simultaneous identification of such compounds with pharmacological activities can more comprehensively and completely reflect the quality of the Chinese medicine A. barbadense Mill., and has important significance for the quality evaluation of the Chinese medicine A. barbadense Mill.

[0082] Example 2 This example relates to the methodology investigation of the method of the present application

[0083] Example 2.1 Precision

[0084] (1) Preparation of the test solution: same as Example 1.

[0085] (2) Preparation of the control solution: same as Example 1.

[0086] (3) HPLC-DAD-ESI-IT-TOF-MS n Detection: same as Example 1, except that the test solution and the control solution are both detected for 6 times continuously. The detection results show that 168 chemical components are detected for 6 times continuously, and 78 chemical components are identified. It can be known that the precision of the instrument used in the method of the present application is good.

[0087] Example 2.2 Stability

[0088] (1) Preparation of the test solution: same as Example 1, except that 6 test solutions are prepared, and are injected after 0h, 3h, 6h, 12h, 18h and 24h respectively.

[0089] (2) Preparation of the control solution: same as Example 1, except that 6 control solutions are prepared, and are injected after 0h, 3h, 6h, 12h, 18h and 24h respectively.

[0090] (3) HPLC-DAD-ESI-IT-TOF-MS n Detection: same as Example 1. The detection results show that 168 chemical components are detected after 24h, and 78 chemical components are identified. It can be known that the test solution in the method of the present application has good stability within 24h.

[0091] Example 2.3 Reproducibility

[0092] (1) Preparation of the test solution: same as Example 1, except that 6 test solutions are prepared.

[0093] (2) Preparation of the control solution: same as Example 1, except that 6 control solutions are prepared.

[0094] (3) HPLC-DAD-ESI-IT-TOF-MS n Detection: same as Example 1. The detection results show that 168 chemical components are detected in the 6 test solutions, and 78 chemical components are identified. It can be known that the method of the present application has good reproducibility.

[0095] Comparative Example 1

[0096] This comparative example is substantially the same as Example 1, except that the relative collision induced dissociation energy is 20%. The detection results show that Comparative Example 1 cannot simultaneously identify 78 components in the Chinese herbal medicine Aloe barbadensis Miller.

[0097] Comparative Example 2

[0098] This comparative example is substantially the same as Example 1, except that the heating block temperature and the curved desolvation tube temperature are 250°C. The detection results show that Comparative Example 2 cannot simultaneously identify 78 components in the Chinese herbal medicine Aloe barbadensis Miller.

[0099] Comparative Example 3

[0100] This comparative example is substantially the same as Example 1, except that the gradient elution degree is different. The gradient elution procedure used in this comparative example is as follows:

[0101] Table 3 Gradient elution procedure of Comparative Example 3

[0102] Time Mobile phase A / vol% Mobile phase B / vol% 0 - 80 min 90％～75％ 10％～25％ 80 - 120 min 75％～70％ 25％～30％ 120 - 150 min 70％～50％ 30％～50％ 150 - 170 min 50％～0％ 50％～100％ 170 - 175 min 0％ 100％

[0103] The detection results show that Comparative Example 3 cannot simultaneously identify 78 components in the Chinese herbal medicine Aloe barbadensis Miller.

[0104] Comparative Example 4

[0105] This comparative example is substantially the same as Example 1, except that the gradient elution degree is different. The gradient elution procedure used in this comparative example is as follows:

[0106] Table 4 Gradient elution procedure of Comparative Example 4

[0107]

[0108]

[0109] The detection results show that Comparative Example 4 cannot simultaneously identify 78 components in the Chinese herbal medicine Aloe barbadensis Miller.

Claims

1. A method for simultaneously identifying 78 components in a traditional Chinese medicine, Aloe barbadensis Miller, characterized in that: Comprise the following steps (1) Preparation of test solution: take the aloe barbadensis miller powder sample, add methanol and ultrasonic extraction; centrifugal take supernatant, filter, filter nitrogen blow, dissolve with methanol to obtain test solution; (2) Preparation of control solution: dissolve the control in methanol to obtain the control solution; the control is aloe new glycoside D, aloe glycoside A, aloe rheum, 7-O-methyl aloe new glycoside A, aloe bitter, rheum emaciatum, altechromone A, rheum-8-O-beta-D-glucopyranoside and aloe ning A; (3) Detection: the test solution of step (1) and the control solution of step (2) were injected into HPLC-DAD-ESI-IT-TOF-MS respectively n for detection; the ion source was an electrospray ion source; the detection mode was negative ion mode; the mass scan range was m / z 50-1000 (MS), m / z 50-1000 (MS2 and MS3); The relative collision induced dissociation energy is 50%; the heating block temperature is 200 DEG C; the curved desolvation tube temperature is 200 DEG C; the detection voltage is 1.70kV; the interface voltage is (+) 4.5kV, (-) 3.5kV; the atomization gas flow rate is 1.5L / min; the chromatographic column is Phenomenex Gemini C18 chromatographic column; the guard column is Phenomenex Security Guard; The elution mode of the HPLC-DAD is gradient elution, the mobile phase of the gradient elution includes mobile phase A and mobile phase B; the mobile phase A is pure water, the mobile phase B is acetonitrile; the sum of the volume fraction of the mobile phase A and the mobile phase B is 100%; the elution procedure of the gradient elution is as follows 。 2. The method of claim 1, wherein: In step (1), The ultrasonic temperature is 20-50 DEG C, and the ultrasonic time is 20-60 min; and / or The filtration is microfiltration membrane filtration, and the pore size of the microfiltration membrane is 0.20-0.22 mu m; and / or The concentration of the test solution is 5-20 mg / mL.

3. The method of claim 1, wherein: In step (2), The concentration of the control solution is 0.2-2 mg / mL.

4. The method of claim 1, wherein: In step (3), The chromatographic column size is 250 mm x 4.6 mm, and the chromatographic column filler particle size is 5 mu m; and / or The guard column size is 4 mm x 3.0 mm, and the guard column filler particle size is 5.0 mu m; and / or The column temperature is 35 DEG C; and / or The injection volume is 10 mu L; and / or The wavelength is 190-400 nm; and / or The flow rate is 0.7-1.3 mL / min.

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